micro RNAs (miRNAs), which are related to small interfering RNAs (siRNAs), are evolutionarily conserved, non-coding, small RNAs (21-25 nt) involved in post-transcriptional gene regulation. To further understand the biogenesis and functions of these RNAs and to develop new siRNA expression systems for a variety of genetic and therapeutic applications, optimized purification and detection methods are required.

We report here that the detection of tiny RNAs by hybridization in solution is a faster and more sensitive alternative to standard Northern blotting techniques. While detection of miRNA on solid support as in Northern- or dot-blotting protocols typically requires several µg of total RNA, quantitative analyses can be performed in solution with as little as 50 ng of total RNA. Such solution hybridization allows detection of amol (10-18 mol) amounts of target RNA. Another advantage of this approach is the potential to simultaneously detect in the same experimental sample several tiny RNA of the same size or both tiny RNA and long RNA species (e.g. siRNA and target mRNA).

Using both solid support and solution hybridization, we also show dramatic variations in small RNA recovery using different standard RNA purification techniques. Even small RNAs commonly used as a loading control (e.g. 5S rRNA) are not quantitatively recovered, and these variations can fuel misinterpretations or lead to incorrect conclusions about miRNA expression patterns. We present improved procedures to efficiently purify representative total RNA populations or isolate control fractions specifically enriched or depleted in small RNA species.

Finally, the specificity and sensitivity of these new tools is illustrated by various studies aimed at quantifying siRNA or miRNA expression levels, correlating siRNA expression and target mRNA knockdown using various siRNA expression systems or analyzing variation of miRNA expression and distribution in different tissues.

A Sensitive, Specific and Versatile Detection Assay

Figure 1. Sensitive and Specific Detection of Tiny RNAs. Because solution hybridization is known to be more sensitive than hybridization on solid support, we investigated the solution-based RNase protection assay for its ability to detect small RNAs. (A) The indicated amounts of a 19 nt antisense GAPDH siRNA were spiked into 4 µg of yeast RNA and detected with a 29 nt long radiolabeled probe prepared by in vitro transcription (IVT). The probe carries a 10 nt sequence at its 5' end that is not complementary to GAPDH siRNA and is cleavable by RNases. After incubation at 42°C for 15 hours, reactions were treated with RNases A and T1 for 30 min at 37°C. Protected fragments (19 nt) were recovered by precipitation and analyzed on a 15% denaturing polyacrylamide gel. (B) Same experiment with 200 attomole of sense or antisense GAPDH siRNA and radiolabeled probes specific for either strand. The antisense mut probe carries 3 mismatch mutations (UGU to GUG) corresponding to nucleotides 9 to 11 of the antisense strand of GAPDH siRNA sequence. No protected fragment was detected in the absence of the appropriate target RNA or with the mutated probe. (C) mir-16 and mir-22 (22 nt) expression was analyzed with 1 µg of FirstChoice™ Total RNA from mouse kidney and 32 nt long radiolabeled probes. mir-16 mut probe (32 nt) carries 3 mismatch mutations (ACG to CGA) corresponding to nucleotides 9 to 11 of the miR-16 miRNA sequence. The mir-16 +4 probe (36 nt) carries 4 additional A residues between the 22 nt sequence specific for miR-16 and the 10 nt leader sequence, producing a 26 nt long protected fragment. This probe design allows simultaneous detection of miR-16 and miR-22 in the same experimental sample.

Figure 2. Examples of Solution Hybridization Assays. (A) Versatility of the assay. The indicated single or multiple target RNAs were detected in 500, 250, 100 and 50 ng of total RNA from mouse tissues (3.5 hours exposure) or HeLa cells (6 hours exposure). The probe specific for GAPDH mRNA (39 nt) produces a 29 nt long protected fragment with the same specific activity as the mir-16 protected fragment, allowing direct comparison of miR-16 miRNA and GAPDH mRNA expression levels. GAPDH siRNA expression in HeLa cells was analyzed 3 days after transfection with pSilencer™ 2.0-U6 GAPDH. (B) Comparison between Northern blot and solution hybridization. Various small RNA expression levels were analyzed in 0.5, 1, 2 or 4 µg of FirstChoice Total RNA from mouse kidney using the indicated DNA or RNA probes prepared by IVT or by 5' end labeling reaction with T4 PNK. Detection of miR-16 miRNA was ~100 fold more sensitive with solution hybridization as compared to Northern blot. This experiment shows that 5' end labeled RNA probes can also be used to detect miR-16 miRNA by solution hybridization (with ~2 fold reduction in sensitivity). In this case, the probe carries a 6 nt additional sequence at its 3' end that is not complementary to miR-16 and is cleavable by RNases, enabling the distinction of full length probe from protected fragment.

Application: Analysis of miRNA Expression

Figure 3. Detection of miRNAs Across Different Tissues. (A) miR-16, miR-20 and miR-22 miRNAs were detected in 1 µg of FirstChoice Total RNA from 10 different mouse tissues. The same differential expression of miR-16 across tissues was observed by Northern blot analysis (2 days exposure) or by hybridization in solution (2 hr exposure). miR-20 is expressed at levels that are not detectable by Northern blot and was previously reported as not expressed in mouse kidney (Lagos-Quintana et al., Science vol 294, 2001). However miR-20 was easily detected by solution hybridization (14 hours exposure). As a control, the same RNA samples (1 µg) were also resolved on a 1.2 % denaturing agarose gel and U1 snRNA, ß-actin and GAPDH mRNA expression was analyzed by Northern blot using the NorthernMax®-Gly Kit. (B) miR-200b, miR-16 and miR-22 miRNAs were detected in 1 µg of FirstChoice™ Total RNA from 12 different human tissues. miR-200b and let-7 expression was also analyzed by Northern blot (4 µg of total RNA). While miR-200b was previously reported to be expressed only in human lung (Grad et al., Molecular Cell vol 11, 2003), we could detect it also in colon, kidney, pancreas, prostate and thymus using both Northern blot and solution hybridization. We attribute this discrepancy to the use of FirstChoice Total RNA validated for miRNA research. Other commercial total RNA preparations may lack small RNA species such as snRNAs, 5S rRNA, tRNAs and miRNAs (see also Fig. 6). 5S and 5.8S rRNAs were used as loading controls.

Figure 4. miR-16 and miR-22 Expression in Mouse Brain. Little or no data are available about miRNA distribution within cells or tissues. This lack of information mainly stems from the purported difficulties in using very small probes for in situ hybridization studies. We analyzed miR-16 and miR-22 distribution in coronal mouse forebrain sections using nonisotopically labeled 32 nt long probes. miR-16 and miR-22 miRNAs were found widely distributed in cortex layers 2 and 3 (Panels B and E). Consistent with this result, both miRNAs were also detected in mouse brain total RNA by solution hybridization using the same radiolabeled probes (see Fig. 3A). However, differences between the miR-16 and miR-22 expression patterns in the brain were noted. For example, very few miR-16 positive cells were observed in the head of the caudate nucleus (Panels C and F). The staining pattern in this area indicated a cytoplasmic subcellular localization for both miRNAs. As a control we also analyzed the distribution of VIP mRNA in mouse cortex. The same cell-specific expression of VIP mRNA was observed with a 1.5 kb probe (A) or with a 22 nt long probe (D), showing that small probes yield specific signal. In situ hybridized cells in the mouse brain cortex (A,B,D,E; 20X magnification) and the head of the caudate nucleus (C,F; 40X magnification) are indicated by arrows.

Figure 5. Analysis of GAPDH siRNA Expression and mRNA Knockdown. For the development of improved siRNA delivery systems and siRNA expression vectors, particularly those that permit targeted delivery or tissue specific expression, one needs to monitor siRNA levels, preferably in conjunction with the assessment of target transcripts. The solution hybridization assay permits simultaneous measurement of siRNA expression levels and target mRNA knockdown. (A) HeLa cells were transfected with an siRNA targeting GAPDH or with a pSilencer 2.0-U6 plasmid engineered to express either the GAPDH siRNA or a negative control siRNA (SCR). Three days after transfection, total RNA was isolated and 1 µg was assessed by solution hybridization as described in Fig. 1A. No signal was detected with the negative control plasmid or in cells not transfected. (B) Simultaneous multi target detection with 2 probes specific for GAPDH mRNA or GAPDH siRNA using the same RNA samples described above ("Total RNA") revealed an efficient siRNA-dependent knockdown of GAPDH mRNA (~60%). After removal of the small RNA species from the total RNA samples by passage over a glass fiber filter ("After GFF", see also Fig. 6 and 7), no siRNA was detected but a similar reduction of GAPDH expression was observed. (C) As a control, the same RNA samples (1 µg) were also resolved on a 1.2 % denaturing agarose gel and U1 snRNA, b-actin and GAPDH mRNA expression was analyzed by Northern blot. A similar GAPDH mRNA knockdown was measured while reduction of the U1 signal in the "after GFF" samples confirmed the removal of small RNA species by the GFF treatment.

RNA Isolation and Recovery of Small RNA

Figure 6. Differential Recovery of Small RNAs During Total RNA Isolation. (A) Total RNA was isolated from 1x106 HeLa cells using three different techniques: monophasic phenol/chaotropic extraction (MPCE), binding on glass fiber filter in guanidinium solution (GFF) or double phenol/guanidinium extraction (DPGE). One µg of purified RNA was resolved on a 1.2% denaturing agarose gel (top left panel) or 15% denaturing polyacrylamide gel (bottom left panel). The indicated mRNAs or small RNAs were detected by Northern blot or solution hybridization (right panels). While mRNAs and 5.8S rRNA were efficiently recovered with all 3 methods, other small RNAs such as U1 snRNA, 5S rRNA and several miRNAs were partially or completely depleted in the total RNA isolated with glass fiber filter (GFF). Thus, for experiments aimed at analyzing miRNA expression paterns, we recommend using both 5S and 5.8S rRNAs as loading controls or probing for another constitutively expressed small RNA such as U1 snRNA (see also Fig. 3; note that FirstChoice™ Mouse and Human Total RNA contain small RNA species and have been validated for miRNA research). (B) To confirm our results, 20 ug of FirstChoice Mouse Kidney Total RNA was either bound and eluted from a GFF or precipitated with 0.5 M NH4OAc and 3 volumes of EtOH. One µg of the untreated or recovered RNAs were compared by gel analysis, Northern blot or solution hybridization as described in (A).

Figure 7. Improved RNA Isolation Procedure for Efficient miRNA Recovery. We developed a rapid glass fiber filter-based procedure allowing isolation of total RNA together with small RNA species, or the preparation of fractions specifically enriched in RNAs smaller than ~200 nt. (A) Total RNA was isolated from the same mouse liver lysate using a double phenol/guanidinium extraction (DPGE) or the new GFF procedure in triplicate (miRNA 1 to 3). The experiment was performed with two different mouse liver lysates (Liver 1 or 2). One µg of each sample RNA was analyzed on a denaturing 15% polyacrylamide gel. Staining with ethidium bromide revealed an efficient recovery of small rRNAs and tRNAs with both procedures. (B) RNAs from the same gel were transferred to a membrane and probed for U2 snRNA and let-7 miRNA. The relative amount of small RNA in each lane was quantified with a phosphorimager. The graph shows the percentage of recovery respectively to the DPGE prep. (C) Total RNA was isolated with the DPGE protocol from one half of 4 different lysates prepared from mouse brain, heart, kidney or liver. The other half of each lysate was used to isolate small RNA species using the novel GFF enrichment procedure. One µg of each RNA sample, as well as the depleted fraction complementary of the enriched fraction, was analyzed on a denaturing 15% polyacrylamide gel stained with ethidium bromide. Most of the small rRNAs and tRNAs were found in the enriched fraction. (D) The relative abundance of U2 snRNA and let-7 miRNA in each lane was analyzed as in (B). The graph shows a significant enrichment of U2 and let-7 with all four tissues tested (310 to 770%).

Conclusion

Solution hybridization assay is faster and ~100 fold more sensitive than Northern blot

Solution hybridization assay allows detection of several miRNAs or both small RNA and longer RNA species in the same experimental sample